Mast cells orchestrate the recruitment of inflammatory cells and initiate and perpetuate allergic responses through their ability to release a wide array of inflammatory mediators, including histamine and other preformed mediators, de novo synthesized arachidonic acid metabolites (leukotrienes and prostaglandins), and numerous proinflammatory cytokines and chemokines. All have been shown to play important roles in the pathogenesis of allergic disorders, such as anaphylaxis and asthma, and their exacerbation. Our recent studies have begun to implicate the potent sphingolipid metabolites, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) and the kinases that produce them, sphingosine kinases (SphK1 and SphK2) and ceramide kinase (CerK), respectively, in regulation of degranulation of mast cells and their secretion of chemokines and cytokines, and eicosanoid synthesis (particularly PGD2 and CysLT). This proposal is aimed at enhancing understanding of the roles of these sphingolipid metabolites and the enzymes that regulate their levels in human mast cell functions in allergic responses and anaphylaxis. It is our hypothesis that SphK1 (and possibly SphK2) are novel targets for the treatment of allergic responses in which mast cells play pivotal roles. In Aim 1, we will substantiate the involvement of S1P and C1P and the enzymes that regulate their levels in amplifying and perpetuating allergic responses of human mast cells. In Aim 2, we will determine the effectiveness of novel inhibitors (targeting the kinases producing S1P and C1P) and FTY720 analogues that block eicosanoid production on human mast cell functions. Aim 3 is focused on determining how S1P is secreted by human mast cells. Finally, in Aim 4, we will determine the effectiveness of the novel inhibitors developed in Aim 2 in alleviation of cutaneous and systemic anaphylaxis and airway hyper-responsiveness in murine models. These studies will further our understanding of the critical role of S1P in orchestrating human mast cell functions and immune reactions, providing the basis for development of therapeutic agents that target the enzymes that regulate its levels and "pave the way" for the development of potent and specific drugs that potentially could be useful for treating various severe human immune responses, including anaphylaxis and asthma. Mast cells orchestrate the recruitment of inflammatory cells and initiate and perpetuate allergic responses. Public Health Relevance: This proposal is focused on the role of bioactive sphingolipid metabolites in regulating functions of human mast cells. Sphingolipid signaling is central to human allergic diseases and potentially amenable to therapeutic targeting. The proposed studies hold much promise for treatment of allergic disorders.